TNF-α dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation

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Abstract
Expression of TNF-α, a pleiotropic cytokine, is elevated during stroke and cerebral ischemia. TNF-α regulates arterial diameter, although mechanisms mediating this effect are unclear. In the present study, we tested the hypothesis that TNF-α regulates the diameter of resistance-sized (∼150-μm diameter) cerebral arteries by modulating local and global intracellular Ca2+ signals in smooth muscle cells. Laser-scanning confocal imaging revealed that TNF-α increased Ca2+ spark and Ca2+ wave frequency but reduced global intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle cells of intact arteries. TNF-α elevated reactive oxygen species (ROS) in smooth muscle cells of intact arteries, and this increase was prevented by apocynin or diphenyleneiodonium (DPI), both of which are NAD(P)H oxidase blockers, but was unaffected by inhibitors of other ROS-generating enzymes. In voltage-clamped (−40 mV) cells, TNF-α increased the frequency and amplitude of Ca2+ spark-induced, large-conductance, Ca2+-activated K+ (KCa) channel transients ∼1.7- and ∼1.4-fold, respectively. TNF-α-induced transient KCa current activation was reversed by apocynin or by Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a membrane-permeant antioxidant, and was prevented by intracellular dialysis of catalase. TNF-α induced reversible and similar amplitude dilations in either endothelium-intact or endothelium-denuded pressurized (60 mmHg) cerebral arteries. MnTMPyP, thapsigargin, a sarcoplasmic reticulum Ca2+-ATPase blocker that inhibits Ca2+ sparks, and iberiotoxin, a KCa channel blocker, reduced TNF-α-induced vasodilations to between 15 and 33% of control. In summary, our data indicate that TNF-α activates NAD(P)H oxidase, resulting in an increase in intracellular H2O2 that stimulates Ca2+ sparks and transient KCa currents, leading to a reduction in global [Ca2+]i, and vasodilation.